PROJECT SUMMARY/ABSTRACT We are dedicated to deciphering the molecular mechanisms central to two essential processes: transcriptional regulation of gene expression and chromosome segregation by the microtubule (MT) cytoskeleton during cell division. We are using cryo-EM to visualize the molecular players critical to those processes. Gene transcription is a complex task, critical for growth and survival. While initiation is the most regulated step in transcription, and its fine-tuning can produce organism-wide changes in gene expression profiles, a mechanistic understanding lags behind due to the complexity of the molecular machinery involved. In addition to the RNA polymerase II (Pol II), general transcription factors (GTFs: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH)) are required to find the transcription start site (TTS) and to melt and load the DNA onto Pol II. TFIID (~ 1 MDa) is required for binding to different core promoter sequences and for activated transcription. TFIIH (~450 kDa) is essential for promoter melting and the phosphorylation of Pol II needed to clear the promoter, as well as for DNA repair. Structural analysis of the eukaryotic transcriptional machinery is extremely difficult due to its scarcity, poor stability, and its intrinsic flexibility. My lab has made substantial progress in describing the architecture and DNA interactions of human TFIID, and visualizing the human transcription preinitiation complex (PIC) of GTFs in different states, uniquely contributing to establishing a structural framework for the transcription initiation process. We are now well poised to make further contributions to this field. We will define the atomic structures of TFIID and TFIIH, which lag behind, and build complexity by adding gene-specific transcription factors to our human PICs in order to provide insights into the structural basis of transcriptional regulation. Cell division is a complex, highly regulated process in which the microtubule (MT) cytoskeleton plays a central role, serving as energy source for dramatic chromosomal movements and acting as a scaffold that facilitates molecular encounters at the right time and place. Essential for MT function is dynamic instability, a property that can be both regulated and utilized for cellular work. The MT is built by the self-assembly of ab-tubulin dimers and MT dynamics are due to the coupling of the assembly process to GTP hydrolysis in b-tubulin. Anticancer drugs like taxol stop cell division by interfering with MT dynamics, while many MT cellular partners modulate or utilize dynamic instability to carry out specific functions. By characterizing in atomic detail the conformational changes in MTs that accompany GTP hydrolysis, my lab has shed unique light into the structural basis of MT dynamic instability. We have also visualized the binding site and effect of anticancer drugs on MTs, and started to define how cellular factors interact with the MT surface, potentially affecting its structure and stability. Our main effort now is to extend our knowledge on the binding and effect of mitotic factors that regulate MT dynamics and organization, and shed mechanistic light into a fundamental process for the eukaryotic cell that will contribute to the improvement/development of anticancer agents that interfere with mitosis.